Recent successes in targeted immune and cell-based therapies have driven new directions for pharmaceutical research. With the rise of these new therapies there is an unfilled need for companion diagnostics to assess patients’ potential for therapeutic response. Targeted nanomaterials have been widely investigated to fill this niche; however, in contrast to small molecule or peptide-based targeted agents, binding affinities are not reported for nanomaterials, and to date there has been no standard, quantitative measure for the interaction of targeted nanoparticle agents with their targets. Without a standard measure, accurate comparisons between systems and optimization of targeting behavior are challenging. Here, we demonstrate a method for quantitative assessment of the binding affinity for targeted nanoparticles to cell surface receptors in living systems and apply it to optimize the development of a novel targeted nanoprobe for imaging vulnerable atherosclerotic plaques. In this work, we developed sulfated dextran-coated iron oxide nanoparticles with specific targeting to macrophages, a cell type whose density strongly correlates with plaque vulnerability. Detailed quantitative, in vitro characterizations of 111In3+ radiolabeled probes show high-affinity binding to the macrophage scavenger receptor A (SR-A). Cell uptake studies illustrate that higher surface sulfation levels result in much higher uptake efficiency by macrophages. We use a modified Scatchard analysis to quantitatively describe nanoparticle binding to targeted receptors. This characterization represents a potential new standard metric for targeted nanomaterials.

Despite advances in diagnosis and management, cardiovascular diseases (CVDs) remain 22 the leading cause of death in the US. Atherosclerosis is the most common form of CVD and the 23 vulnerability of atherosclerotic plaques to rupture is a primary determinant for risk of catastrophic 24 ischemic events. Current imaging of atherosclerotic disease focuses on assessing plaque size and 25 the degree of luminal stenosis, which are not good predictors of plaque stability. Functional 26 methods to identify biomarkers of inflammation in plaques could facilitate assessment of plaque 27instability to allow early intervention. In this study, we validate the use of a purpose-built, 28 magnetic resonance imaging (MRI)-compatible positron emission tomography (PET) insert for 29 multimodal, molecular imaging of vulnerable plaques in mice. We illustrate the application of PET 30 to screen for inflamed regions to guide the application of MRI. Molecular MRI visualizes regions 31 of vascular inflammation and is coupled with anatomical MRI to generate detailed maps of the 32 inflammatory marker within the context of an individual vessel. As a testbed for this imaging 33 methodology, we developed a multimodal, iron oxide nanoparticle (NP) targeting vascular cell 34 adhesion molecule-1 (VCAM-1) for simultaneous PET/MRI of vascular inflammation performed 35 on a mouse carotid ligation model. In vitro cell studies confirmed that the NPs are not cytotoxic to 36 liver cells. In vivo simultaneous PET/MRI imaging identified regions of inflammation.37 Three-dimensional rendering of the MRI data facilitated high-resolution visualization of patterns 38 of inflammation along the injured vessel. Histology validated the co-localization of the NPs with 39 VCAM-1 expression at sites of induced inflammation. The results of this work validate the utility 40 of the simultaneous PET/MR insert as a research tool for small animals and lays groundwork to 41 further advance the potential clinical utility of integrated imaging systems.42 43 vulnerable plaque; cardiovascular imaging; vessel wall; VCAM. 44 45 1. Introduction localized inflammatory response can lead to the development of "vulnerable" plaques that are 51 prone to rupture and cause downstream vascular occlusion.[3] Imaging can play a role in 52 identifying patients with vascular lesions susceptible to acute cardiovascular events, who may be 53 amenable to treatment with anti-inflammatories or interventional procedures. Recent literature has 54 shown that plaque lesion composition, particularly the presence of inflammatory markers and 55 immune cells, as opposed to the degree of vessel stenosis, is a better predictor of patient mortality 56 and morbidity; and assessment of plaque inflammation is an excellent target for noninvasive 57 imaging.[4] However, current clinical imaging techniques seldom provide specific information 58 about inflammation.59 Current clinical imaging techniques such as coronary angiography, vascular ultrasound and 60 computed tomography focus on identifying stenotic diseas...